Resistance is Futile: An Insidious Game of CLUE

https://wiki.mcmaster.ca/LIFESCI_4M03/group_1_presentation_1_-_obesity

As of 2017, an estimated 425 million people are impacted by Diabetes around the world, resulting in a global cost exceeding 825 billion US dollars per year (Shulman, 2018). Does this statistic shock you?

Well if that didn’t do the trick, then how about this one: by 2045 the projection is that there will be roughly 629 million people impacted by Diabetes (a striking 48% increase; Shulman, 2018).

Yet, we throw this term “Diabetes” and “insulin resistance” around quite often, but do we really understand what it means? More importantly, do we really understand the disastrous implications associated with the onset of this disease?

I am sure all of us are quite familiar with the term blood sugar (as most of us throw this term around willy nilly without truly understanding what it means). Blood sugar in essence is the amount of glucose in our blood (pretty straightforward)! Hyperglycemia can then be identified in the clinical setting, thereby establishing a diagnosis of Diabetes (it is a bit more complicated than this, but for simplicity I will keep it at this).

So how does this differ from what happens in a metabolically healthy individual? Well in a metabolically healthy individual, upon consumption of glucose, a subset of cells located in your pancreas (beta cells of the Islet of Langerhans) are stimulated to release insulin. One of insulin’s main roles is to then enhance the uptake of glucose via your skeletal muscle, while simultaneously reducing the production of glucose by your liver (gluconeogenesis). Overall, this results in decreasing your blood sugar levels.

However, what happens when insulin is no longer to propagate these necessary signals? Well, that is precisely what happens in Type 2 Diabetes Mellitus. Insulin is no longer able to elicit the proper signals to enhance glucose uptake in the skeletal muscle and reduce glucose production in the liver. Accordingly, because your body realizes that insulin is no longer doing its job properly (reducing blood sugar levels), a signal is sent to the beta cells of your pancreas to work overtime to hopefully make enough insulin to get the job done. Unfortunately, this causes severe damage to your pancreas, and may even lead to permanent dysregulation of insulin secretion. Ultimately, this inability of insulin to properly propagate its signal is called insulin resistance (a term I am sure most of you are familiar with), and ultimately leads to the elevated blood sugar levels (hyperglycemia) that we associate with Diabetes.

So now that we have a bit of a better sense of what exactly hyperglycemia and insulin resistance are and how they relate to Diabetes, we need to investigate what causes this insulin resistance.

To begin, we know that ingested carbohydrates have three general fates: 1) glucose can be oxidized (burned) to produce carbon dioxide in the skeletal muscle, 2) glucose can be converted to the metabolite lactate via a process called glycolysis, and 3) glucose can be stored in the liver or muscle as glycogen. So, it has to be one of these three pathways that has gone awry, thereby leading to the downstream effects associated with insulin resistance.

Luckily for us, Dr. Ralph DeFronzo out of the University of Texas San Antonio has us covered. Over the course of a few decades, dating back to the 80s, DeFronzo and his lab were able to determine that the defect in insulin stimulated glucose metabolism seen in Type 2 Diabetes was due to the non-oxidative glucose metabolism (R. DeFronzo, 2014; Defronzo et al., 1981; R. A. DeFronzo, 1988; R. A. DeFronzo et al., 1978, 1985). Hence, if Defronzo and his colleagues determined that this malfunction occurs in the non-oxidative portion, that leaves us with one option for the dysregulation: glucose being stored as glycogen (because the other two options require the oxidation/burning of glucose!) However, this was just a postulation at this point and still needed to be verified in a laboratory setting.

At this point, the baton was handed off to Shulman and colleagues at Yale University. With an “X marks the spot” on glycogen synthesis, Shulman’s work would be easy, right?

Unfortunately, NO! the process of glycogen synthesis is not so simple and there are a lot of moving parts. Accordingly, Shulman and colleagues conducted several elegant experiments to determine which exact part of glycogen synthesis has gone awry in Type 2 Diabetics. Using the techniques of a hyperglycemic — hyperinsulinemic clamp and carbon nuclear magnetic spectroscopy (13C NMR) Shulman set out to determine if glycogen synthesis was our main culprit in insulin resistance. Although these techniques sound scary, they are quite straightforward. Shulman added a radiolabel to one of the carbon atoms of glucose that could be tracked using the 13C NMR machine once the glucose was ingested. Remember from previous discussions that glucose is a molecule made up of 6 carbon atoms, so Shulman would label one carbon in each molecule of glucose so he could track it (it is a lot more complicated than that, but this is the barebones needed for comprehension). Shulman would then infuse the glucose into 5 diabetic and 6 weight-matched healthy patients and coordinated this glucose infusion with insulin infusion (This is our hyperglycemic-hyperinsulinemic clamp). The researchers then analyzed the rate of incorporation of the labeled glucose into muscle glycogen (he looked specifically at the gastrocnemius muscles)using his NMR technique.

Without throwing meaningless rate values at you, Shulman and colleagues did in fact conclude that glucose conversion into glycogen was significantly decreased in Diabetics when compared to controls. Hence, DeFronzo was correct in that muscle glycogen synthesis is the principal pathway of glucose disposal in normal and diabetic individuals, and that defects in this specific pathway were the culprit leading to insulin resistance (G. I. Shulman et al., 1990). Now the question remains: Where in the muscle glycogen synthesis pathway is the defect?

https://www.youtube.com/watch?v=mrpQeRY7jXU

Above you can see an image of the normal pathway to convert glucose into glycogen. Now yes there are some complex enzymatic names here, but I will outline it all very simply (so you don’t get caught in the weeds). When insulin is released from your beta cells in the Islet of Langerhans, a big cascade of signals occur that ultimately results with that yellow guy, GLUT-4, getting moved from inside the cell to the cell membrane (this is called translocation). This is key, because GLUT-4 is the door that glucose uses to get inside our muscle cells. Although not as simple as this, you can think “no GLUT-4, no entry.” If glucose successfully makes its way inside the muscle cell, an enzyme called hexokinase turns glucose into a different molecule called glucose-6-phopshate (this is just normal glucose with a phosphate group attached). Glucose-6-phopshate is then converted into a molecule called UDP-glucose (after several steps) which is then finally converted to glycogen via the enzyme glycogen synthase. Glycogen is then able to be stored in the muscle (glycogen can also be stored in the liver, but for the purposes of this example we are using the skeletal muscle).

So as I mentioned before, even though we narrowed it down to a defect in the muscle glycogen synthesis pathway, that was not enough. Accordingly, within the glycogen synthesis pathway, we have three specific culprits: 1) GLUT-4, 2) Hexokinase, and 3) Glycogen synthase

So think of GLUT-4, hexokinase, and glycogen synthase as potential bottlenecks. If they are not working properly, there will be a buildup of the molecules prior to that step, and a reduction in the molecules after that step. Hence the following hypotheses:

1) If Glycogen synthase is dysregulated in this pathway, then we should see decreased glycogen and a buildup of UDP-Glucose, glucose-6-phosphate, and intracellular glucose

2) If hexokinase is dysregulated in this pathway, we should see decreased glucose-6-phosphate, decreased UDP-glucose, decreased glycogen, and increased intracellular glucose

3) If GLUT-4 is dysregulated then we should see increased plasma glucose and a reduction in the other key players

Once again using NMR, researchers found that both intracellular glucose and glucose-6-phosphate levels were decreased in Diabetics. Thus, it would appear that there is a malfunction in the GLUT-4 (Cline et al., 1999).

Wow so problem solved! Yeah, not exactly. See the thing with science is that anytime you answer one question, two more questions arise. Even though we have narrowed it down to GLUT-4 at this point, we still don’t know what exactly about GLUT-4 is going wrong. For instance, is GLUT-4 not getting the proper signal to move to the cell membrane, thereby not allowing glucose into the cell? Or, maybe GLUT-4 still gets the signal, makes it way to the cell membrane, but for some reason will not open to allow glucose into the cell? Tricky, tricky. And what exactly is causing this insulin resistance to begin with?

Well it turns out, we for sure have the answer to that last question. We know that intramyocellular lipids (fat that has gotten inside the muscle cell) are a tremendous indicator of whole-body insulin sensitivity (Krssak et al., 1999). Therefore, the more fat inside one’s muscle cell, the less insulin resistant they are (although there is something called the Athlete’s paradox…that is a different and highly complex argument, however. If you are interested about this, reach out).

So, now the puzzle is starting to come together. Maybe fat (or fatty acids when they are broken down) are somehow impeding GLUT-4 from getting to the skeletal muscle membrane or stopping GLUT-4 from allowing glucose entry into the cell. Intriguing postulations, but can we piece this together?

Well I wouldn’t be writing this article if I didn’t think we could :)

https://www.youtube.com/watch?v=mrpQeRY7jXU

This diagram builds upon the one I showed above. So, glucose enters the muscle cell via GLUT-4 and is then converted into our good friend glucose-6-phosphate (G6P) by hexokinase (HK). Then through a process called glycolysis (demonstrated by the enzyme PFK in the diagram), glucose-6-phosphate is eventually converted to pyruvate. Finally, pyruvate is converted to the key metabolite, Acetyl CoA, by the enzyme pyruvate dehydrogenase (PDH). Acetyl CoA can then be used for various crucial biochemical/metabolic pathways in the cell.

As per above, there are several potential bottleneck points, and we have to ask the question: what is decreased when we expose this cell to increased amounts of fatty acids?

Well, it turns out that both G6P (Roden et al., 1996) and glucose (Dresner et al., 1999) are decreased when we expose human cells to fatty acids. What does this tell us? It tells us that free fatty acids must block GLUT-4, thereby inducing insulin resistance.

I feel like this is a big game of CLUE. We have narrowed it down to free fatty acids committing the crime on the muscle glycogen synthesis pathway by blocking GLUT-4, but we still are missing one last piece: The weapon! By which mechanism do excess free fatty acids cause this dysregulation

To answer this question, I need to discuss insulin’s mechanism of action, yet this is far too complex and would just muddy the waters for most readers, so I will do my best to simplify.

https://www.youtube.com/watch?v=mrpQeRY7jXU

In this case, insulin is what we call a ligand. Think of a ligand as a very specific key that can really only open a very specific lock, which in this case is something we call a receptor (again this is not completely true but gets the message across). As insulin travels through the bloodstream, it sometimes finds its perfect match and binds to the lock. If the fit is right, the lock is opened thus triggering a huge cascade of events that ultimately results in the movement of GLUT-4 from inside the cell to the cell membrane (as mentioned above). As you can see here in the diagram above, some of our key players in this cascade are Insulin-Receptor Substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI 3-Kinase). So fatty acids could really be impeding the process at any one of these key checkpoints thus impeding GLUT-4’s ability to translocate to the cell membrane.

Now this is where things get dicey. If you search “Insulin Resistance” in PubMed there are over 110,000 citations ranging from implicating ceramides, BCAAs, diacylglycerols, endoplasmic reticulum stress, inflammation, etc…

So we are still missing that final piece…the HOW? I have researched this topic extensively over the last several months at NIH, and have leaned towards what Dr. Shulman has demonstrated in his laboratory more recently (which I will outline below). At the end of the day, insulin resistance has a multifactorial etiology, so there is most likely not one concrete answer. But what I will share below implicates free fatty acids, GLUT-4, and the muscle glycogen synthesis pathway in a way that makes sense in accordance with other research and the biochemical principles at play.

We know that obesity is associated with increased free fatty acid levels (Muoio, 2014; Paniagua, 2016; Smith et al., 2018). Accordingly, the excess circulating free fatty acids throw off the homeostatic balance between fat sorage and fat burning. In this case, more fatty acids are being delivered to the skeletal muscle than can be burned for fuel (remember I told you that we see higher levels of fats inside the muscle cells in insulin resistant individuals). So, what does increased fat in the muscle do? Nothing good, that’s for sure (again if you are an athlete, it is a bit of a different story).

https://www.youtube.com/watch?v=mrpQeRY7jXU

This accumulation of intramyocellular lipids (fat in the skeletal muscle) activates this molecule called diacylglycerol (DAG). DAG then activates a molecule called protein-kinase C-theta which inhibits our good friend IRS-1 (from above). If IRS-1 is inhibited, then it cannot activate PI 3-Kinase which is needed to move GLUT-4 to the cell membrane. Thus, if we cannot get GLUT-4 to the cell membrane, then we cannot get as much glucose inside the cell as we normally could, thereby decreasing muscle glycogen synthesis. This ultimately leads to the dreaded insulin resistance.

This pathway has been shown in various studies and is the one I tend to lean towards(Griffin et al., 2000; Morino et al., 2005; G. I. Shulman, 2014; Szendroedi et al., 2014; Yu et al., 2002).

Wowza, that was a lot, but look at my diagram below to outline everything I went through. Now that we have an idea of what causes insulin resistance, we need to discuss why this is such a problem…a topic that will be covered in my next article!

Cline, G. W., Petersen, K. F., Krssak, M., Shen, J., Hundal, R. S., Trajanoski, Z., Inzucchi, S., Dresner, A., Rothman, D. L., & Shulman, G. I. (1999). Impaired Glucose Transport as a Cause of Decreased Insulin-Stimulated Muscle Glycogen Synthesis in Type 2 Diabetes. In New England Journal of Medicine (Vol. 341, Issue 4, pp. 240–246). https://doi.org/10.1056/nejm199907223410404

DeFronzo, R. (2014). Lilly Lecture 1987 (Vol. 37, Issue June 1988, pp. 667–687).

DeFronzo, R. A. (1988). The triumvirate: β-cell, muscle, liver. A collusion responsible for NIDDM. In Diabetes (Vol. 37, Issue 6, pp. 667–687). https://doi.org/10.2337/diab.37.6.667

DeFronzo, R. A., Ferrannini, E., Hendler, R., Wahren, J., & Felig, P. (1978). Influence of hyperinsulinemia, hyperglycemia, and the route of glucose administration on splanchnic glucose exchange. In Proceedings of the National Academy of Sciences of the United States of America (Vol. 75, Issue 10, pp. 5173–5177). https://doi.org/10.1073/pnas.75.10.5173

DeFronzo, R. A., Gunnarsson, R., Bjorkman, O., Olsson, M., & Wahren, J. (1985). Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. In Journal of Clinical Investigation (Vol. 76, Issue 1, pp. 149–155). https://doi.org/10.1172/JCI111938

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Dresner, A., Laurent, D., Marcucci, M., Griffin, M. E., Dufour, S., Cline, G. W., Slezak, L. A., Andersen, D. K., Hundal, R. S., Rothman, D. L., Petersen, K. F., & Shulman, G. I. (1999). Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. In Journal of Clinical Investigation (Vol. 103, Issue 2, pp. 253–259). https://doi.org/10.1172/JCI5001

Griffin, M. E., Marcucci, M. J., Cline, G. W., Bell, K., Barucci, N., Lee, D., Goodyear, L. J., Kraegen, E. W., White, M. F., & Shulman, G. I. (2000). Free fatty acid-induced insulin resistance is associated with activation of protein kinase C θ and alterations in the insulin signaling cascade. In Diabetes (Vol. 48, Issue 6, pp. 1270–1274). https://doi.org/10.2337/diabetes.48.6.1270

Krssak, M., Falk Petersen, K., Dresner, A., DiPietro, L., Vogel, S. M., Rothman, D. L., Shulman, G. I., & Roden, M. (1999). Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: A 1H NMR spectroscopy study. In Diabetologia (Vol. 42, Issue 1, pp. 113–116). https://doi.org/10.1007/s001250051123

Morino, K., Petersen, K. F., Dufour, S., Befroy, D., Frattini, J., Shatzkes, N., Neschen, S., White, M. F., Bilz, S., Sono, S., Pypaert, M., & Shulman, G. I. (2005). Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. Journal of Clinical Investigation, 115(12), 3587–3593. https://doi.org/10.1172/JCI25151

Muoio, D. M. (2014). Metabolic inflexibility: When mitochondrial indecision leads to metabolic gridlock. In Cell (Vol. 159, Issue 6, pp. 1253–1262). https://doi.org/10.1016/j.cell.2014.11.034

Paniagua, J. A. (2016). Nutrition, insulin resistance and dysfunctional adipose tissue determine the different components of metabolic syndrome. In World Journal of Diabetes (Vol. 7, Issue 19, p. 483). https://doi.org/10.4239/wjd.v7.i19.483

Roden, M., Price, T. B., Perseghin, G., Petersen, K. F., Rothman, D. L., Cline, G. W., & Shulman, G. I. (1996). Mechanism of free fatty acid-induced insulin resistance in humans. Journal of Clinical Investigation, 97(12), 2859–2865. https://doi.org/10.1172/JCI118742

Shulman, G. (2018). (123) 2018 Banting Lecture, Gerald Shulman, Mechanisms of Insulin Resistance_ Obesity, Lipodystrophy, T2DM — YouTube.

Shulman, G. I. (2014). Ectopic Fat in Insulin Resistance, Dyslipidemia, and Cardiometabolic Disease. In New England Journal of Medicine (Vol. 371, Issue 12, pp. 1131–1141). https://doi.org/10.1056/nejmra1011035

Shulman, G. I., Rothman, D. L., Jue, T., Stein, P., DeFronzo, R. A., & Shulman, R. G. (1990). Quantitation of Muscle Glycogen Synthesis in Normal Subjects and Subjects with Non-Insulin-Dependent Diabetes by 13 C Nuclear Magnetic Resonance Spectroscopy . In New England Journal of Medicine (Vol. 322, Issue 4, pp. 223–228). https://doi.org/10.1056/nejm199001253220403

Smith, R. L., Soeters, M. R., Wüst, R. C. I., & Houtkooper, R. H. (2018). Metabolic flexibility as an adaptation to energy resources and requirements in health and disease. In Endocrine Reviews (Vol. 39, Issue 4, pp. 489–517). https://doi.org/10.1210/er.2017-00211

Szendroedi, J., Yoshimura, T., Phielix, E., Koliaki, C., Marcucci, M., Zhang, D., Jelenik, T., Müller, J., Herder, C., Nowotny, P., Shulman, G. I., & Roden, M. (2014). Role of diacylglycerol activation of PKCθ in lipid-induced muscle insulin resistance in humans. In Proceedings of the National Academy of Sciences of the United States of America (Vol. 111, Issue 26, pp. 9597–9602). https://doi.org/10.1073/pnas.1409229111

Yu, C., Chen, Y., Cline, G. W., Zhang, D., Zong, H., Wang, Y., Bergeron, R., Kim, J. K., Cushman, S. W., Cooney, G. J., Atcheson, B., White, M. F., Kraegen, E. W., & Shulman, G. I. (2002). Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. In Journal of Biological Chemistry (Vol. 277, Issue 52, pp. 50230–50236). https://doi.org/10.1074/jbc.M200958200

Post-Baccalaureate research assistant in the Molecular and Clinical Nutrition Lab at the National Institutes of Health